Project Summary/Abstract: Transcriptional and chromatin dysregulation are hallmarks of cancer. In this project, we functionally characterize alterations in the pioneer transcription factor, called FOXA1, that are highly recurrent in hormone-receptor driven cancers and describe an innovative chromatin-based approach to identify and therapeutically target transcriptional pathways that specify cellular identity in cancer. FOXA1 de-compacts chromatin to enable binding of the androgen receptor (AR) to activate the PCa genes. By leveraging an aggregate PCa cohort with 1546 samples, we define three novel structural classes of FOXA1 alterations that notably diverge in clinical incidence, genomic co-alterations, and pathogenic gain-of-functions. Notably, these three alteration classes collectively recur in over 35% of the cases, making FOXA1 the third most-highly altered oncogene in metastatic PCa. Class1 mutations originate in primary PCa without ETS-fusions or SPOP mutations, alter the Wing2 secondary structure within the DNA-binding domain, enable faster nuclear mobility, and strongly activate the AR program of prostate oncogenesis. Contrastingly, class2 mutations are acquired in the metastatic disease, truncate the C-terminal regulatory domain, enable dominant binding to the chromatin, and activate the WNT program of metastasis. Finally, class3 structural variations are enriched in metastatic PCa, cluster within the FOXA1 topological domain, and duplicate the highly conserved enhancer element that drives overexpression of FOXA1. In the F99 phase, I propose to mechanistically elucidate the neomorphic activation of WNT-signaling by the FOXA1 class2 mutants. I hypothesize the C-terminal domain of WT FOXA1 to recruit WNT-repressive cofactors to the chromatin, and thus the C-terminal truncated class2 mutants that dominantly bind to the chromatin to transcriptionally de-repress WNT-signaling. This hypothesis offers a unique opportunity to uncover a novel association between FOXA1 and WNT transcriptional pathways. In the K00 phase, I aim to address the intriguing question of why driver oncogenes are not universally shared across cancer types. As a likely explanation, I postulate the existence of requisite interactions between oncogenic pathways and lineage-specific epigenetic/chromatin architecture in driving the malignant phenotype, akin to FOXA1 or ERG (oncogenes) and AR (lineage-essential gene) in primary PCa. This presents the opportunity of disrupting these essential, lineage-defining pathways in cancer as a promising therapeutic strategy - a concept termed as ?targeting the cancer cell identity.? Overall, our findings till date substantiate FOXA1 as a principal oncogene and a viable therapeutic target in PCa. In the future, I propose to delineate the molecular mechanism of aberrant WNT activation in FOXA1 class2-mutant tumors and propose a novel therapeutic strategy of disrupting the cancer cell identity to improve patient survival. These findings will be presented at research conferences where I can meet with potential K00 mentors, and described in peer-reviewed scientific publications. Training in such a multi-disciplinary environment will provide the foundational knowledge and the essential skills for me to succeed as an independent translational cancer researcher in academia.